Cryo-EM structures of cardiac muscle α-actin mutants M305L and A331P give insights into the structural mechanisms of hypertrophic cardiomyopathy

Fig. 1. Protein purification and cryo-EM analysis of cardiac muscle α-actin mutants M305L and A331P. (A) Representative purifications of native (WT) cardiac muscle α-actin and mutants M305L and A331P resolved on an SDS-PAGE gel. The proteins have an apparent molecular weight of 42 kDa. (B) Multiple sequence alignment of human actin isoforms. Numbering according to the sequence of mature cardiac muscle α-actin. Mutations M305L and A331P in the cardiac muscle α-actin sequence are highlighted in red. Sequence deviations from the consensus sequence between actin isoforms are highlighted in dark grey. Reported mutations in residues in homologous positions to cardiac muscle α-actin M305L and A331 are highlighted in orange. The three adjacent proline residues in A331P are indicated with a red box. Cardiac α, cardiac muscle α-actin; M305L, cardiac muscle α-actin harboring mutation M305L; A331P, cardiac muscle α-actin harboring mutation A331P; Skeletal α, skeletal muscle α-actin; Smooth, smooth muscle α-actin; Cyto β, cytoplasmic β-actin; Cyto γ, cytoplasmic γ-actin; Smooth γ, smooth muscle γ-actin. Capital letters in the consensus sequence indicate identical residues, small letters indicate…

Fig. 2. Overall architecture of cardiac muscle α-actin mutants M305L and A331P. (A) Cryo-EM reconstruction of M305L. Four protomers, indicated as a, a+1, a-1, and a-2, are shown. The barbed (+) end and the pointed (-) end are indicated. (B) Model of M305L (cartoon representation) fitted in the electron density map (transparent surface representation). Individual protomers are color coded according to (A). (C) Model of M305L in cartoon representation. The mutated residue L305 is shown in sphere representation. The bound ADP and Mg2+ are shown in stick and sphere representation, respectively. Individual protomers are color coded according to (A). (D) Position of M305L (blue) in the actin protomer (cartoon representation) with respect to the nucleotide binding cleft, the D-loop, the barbed end groove, and the bound ADP (stick representation) and Mg2+ (sphere). Actin subdomains are indicated. Cryo-EM densities are shown in transparent surface representation. (E) Cryo-EM reconstruction of A331P. Four protomers are shown. (F) Model of A331P (cartoon representation) fitted in the electron density map (transparent surface representation). Individual protomers are color coded according to (E).…

Fig. 3. Interprotomer interactions in HCM mutants M305L and A331P. (A) Interprotomer contacts in native cardiac muscle α-actin (PDB ID: 8DMY). (B) Interprotomer contacts in M305L. (C) Interprotomer contacts in A331P. Note that in (A)-(C) labels represent the approximate location of the interprotomer interface in the actin filament. Individual protomers are color coded. (D) Plot of the BSA between neighboring actin protomers in native cardiac muscle α-actin, M305L, and A331P. Significant changes in BSA were only observed along the short pitch helix in M305L and both the long pitch helix and the short pitch helix in A331P compared to native cardiac muscle α-actin.

Fig. 4. Structural features of cardiac muscle α-actin mutant M305L. (A) Structural alterations in the nucleotide binding cleft active site in M305L (cartoon representation). Cryo-EM densities are shown in transparent surface representation. (B) Superimposition of the nucleotide binding cleft active sites of M305L (pink) and native cardiac muscle α-actin (PDB ID: 8DMY, green). (C) Residue H73me in M305L (pink cartoon) fitted in the cryo-EM densities (transparent surface representation). (D) Superimposition of M305L (pink) and native cardiac muscle α-actin (PDB ID: 8DMY, green) shows the altered side chain position of H73me.

Fig. 5. Insights into the nucleotide binding cleft active site of A331P. (A) Structural alterations in the nucleotide binding cleft active site in A331P (cartoon representation). Cryo-EM densities are shown in transparent surface representation. Alternate conformations of the rotamers of K336 are shown. Residue A331P is indicated with a boldface label. (B) Superimposition of the nucleotide binding cleft active sites of A331P (blue) and native cardiac muscle α-actin (PDB ID: 8DMY, green). (C) Close-up view of the nucleotide binding cleft active site in A331P (blue) with cryo-EM densities shown in transparent surface representation. (D) Superimposition of the nucleotide binding cleft active sites of A331P (blue) and native cardiac muscle α-actin (PDB ID: 8DMY, green). Altered residue conformations in the nucleotide binding cleft active sites are indicated with boldface labels.

Fig. 6. Interactions between M305L and A331P with tropomyosin in different functional states. (A) Superimposition of the structure of M305L with tropomyosin in the B-, C-, and M-states (PDB IDs: 5JLF, 5NOG, 5JLH; cartoon representation). Five M305L protomers are shown in surface representation. The mutated residue is shown as blue sphere. (B) Superimposition of the structure of A331P with tropomyosin in the B-, C-, and M-states (PDB IDs: 5JLF, 5NOG, 5JLH; cartoon representation). Five A331P protomers are shown in surface representation. The mutated residue is shown as red sphere.

Fig. 7. Interactions between M305L and A331P with the cardiac myosin motor domain in the rigor state. (A) Superimposition of the structure of M305L with myosin in the rigor state (pink cartoon) (PDB ID: 5JLH). Five M305L protomers are shown in surface representation. The mutated residue is shown as blue sphere. (B) Superimposition of the structure of A331P with myosin in the rigor state (pink cartoon) (PDB ID: 5JLH). Five A331P protomers are shown in surface representation. The mutated residue is shown as red sphere. The white arrow indicates the movement of tropomyosin from the B-state to the M-state.